The goal of this project is to characterize the enzyme(s) that specifically catalyzes the hydrolysis of the sn-2 acetyl residue of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine, platelet- activating factor (PAF). This phospholipid is a potent activator of platelets and nurtrophils, and has marked hemodynamic and hepatic effects. These effects suggest that the concentration of PAF must be precisely regulated to prevent dramatic consequences. This conclusion is supported by animal studies in which an infusion of PAF caused anaphylaxis, shock, and death, as well as in models of anaphylaxis where PAF was shown to appear in blood. Conversely, it has been proposed that PAF is a normal, endogenous antihypertensive compound. These possibilities are not mutually exclusive, but if both obtain, suggest the need for precise control. The products of the acetylhydrolase reaction are without biological activity, which suggests that this enzyme can play a key role in such regulation.
The aims of this project are to complete the characterization of the enzyme that we have purified 25,000-fold from human plasma. We will determine how and where the enzyme is made, and if this process is regulated. The enzyme is part of a lipoprotein particle, ad is found in just two species: LDL, and HDL-with-apoE. The enzyme readily transfer between these two particles, but is only catalytically efficient when in the LDL particle. Thus, we will determine how the enzyme interacts with the two lipoproteins particles in order to understand how environment affects catalysis. We have recently found that in addition to PAF, the purified enzyme will hydrolyze phospholipids when the polyunsaturated fatty acid has been oxidized. It does not hydrolyze long chain fatty acids from phospholipids prior to oxidation, so the enzyme is also a unique phospholipase A2. This type of activity has been suggested to play a key role in the generation of atherogenic lipoproteins that are recognized by the scavenger receptor, and it is thought that it is this receptor that provides the lipid present in the foam cells of fatty streaks. We will determine if the plasma acetylhydrolase is in fact the lipase required for the oxidative modification of LDL. We have discovered an activity in the cytoplasm of erythrocytes that catalyzes the same reaction as the plasma enzyme, but is due to a different enzyme. We will finish our purification of this new activity, compare it to the plasma enzyme that we have previously purified, and will determine what its role is in the turnover of oxidized phospholipids in a cell that is quite susceptible to oxidation. We have identified activities in other cell types that catalyze similar reactions, but differ from either activity. We find that in one of these cell types, monocytes, that the acetlyhydrolase activity is inducible, up to 90-fold, and it is the level of this degradative activity that determines the amount of PAF in the cell. Through the purification and characterization of enzymes that inactivate the potent lipid autacoid PAF, and now that also appear to initiate the destruction of oxidized phospholipids, we will elucidate processes that underlie inflammation shock and atherosclerosis.
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